Pd-l1 and ox40 binding proteins for cancer regulation

ABSTRACT

Provided are monospecific and bispecific proteins that bind specifically to OX40 and/or PD-L1. Exemplary proteins release the inhibition through PD-L1 and stimulate T cell through OX40. Exemplary polyvalent proteins comprise at least one OX40 binding site and at least one PD-L1 binding site. In certain embodiments, the binding sites may be linked through an immunoglobulin constant region. Anti-OX40 and anti-PD-L1 antibodies are also provided.

CROSS - REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. Application No.16/871,799, filed on May 11, 2020, which is a continuation ofInternational Application No. PCT/US2018/067868, filed on Dec. 28, 2018,which claims the benefits of priority of U.S. Provisional ApplicationNo. 62/611,543, filed Dec. 29, 2017, the dislcosures of which areincorporated herein by reference in their entireties.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the XML filecontaining the Sequence Listing is NP-24083-1-US_SEQ_LIST.xml. The sizeof the XML file is 65,027 bytes, and the XML file was created on Mar. 8,2023.

BACKGROUND Field of Invention

The present invention relates to an antibody. More particularly, thepresent invention relates to the antibody for cancer therapy.

Description of Related Art

The two major types of lymphocytes in humans are T (thymus-derived) andB (bone marrow derived. These cells are derived from hematopoietic stemcells in the bone marrow and fetal liver that have committed to thelymphoid development pathway. The progeny of these stem cells followdivergent pathways to mature into either B or T lymphocytes. HumanB-lymphocyte development takes place entirely within the bone marrow. Tcells, on the other hand, develop from immature precursors that leavethe marrow and travel through the bloodstream to the thymus, where theyproliferate and differentiate into mature T lymphocytes.

T Cells

T-cells are the most abundant (about 75% of blood lymphocytes) andpotent immune killer cells. The role of effector T-cells in theanti-tumor immune response is strongly supported by in vitro studies andthe observation that a high infiltration of CD8+ T cells in severaltypes of tumors correlates with a favorable clinical prognostic (Fridmanet al., 2012). The activation of effector naive T-cells requires atleast three complementary signals: (i) TCR-CD3/Ag-MHC interaction withthe assistance of co-receptors (CD4 or CD8); (ii) binding ofco-stimulatory molecules such as CD80 or CD86 to CD28, CD40/CD40L; and(iii) accessory molecules such as cytokines.

Co-stimulation or the provision of two distinct signals to T-cells is awidely accepted model of lymphocyte activation of resting T lymphocytesby antigen-presenting cells (APCs) (Lafferty and Cunningham, 1975). Thismodel further provides for the discrimination of self from non-self andimmune tolerance (Bretscher and Cohn, 1970; Bretscher, 1999; Jenkins andSchwartz, 1987). The primary signal, or antigen specific signal, istransduced through the T-cell receptor (TCR) following recognition offoreign antigen peptide presented in the context of the majorhistocompatibility-complex (MHC). The second or co-stimulatory signal isdelivered to T-cells by co-stimulatory molecules expressed onantigen-presenting cells (APCs), and induce T-cells to promote clonalexpansion, cytokine secretion and effector function (Lenschow et al.,1996). In the absence of costimulation, T-cells can become refractory toantigen stimulation, do not mount an effective immune response, andfurther may result in exhaustion or tolerance to foreign antigens.

Immune Checkpoint Protein: PD-L1 and OX40

Immune checkpoints refer to a group of inhibitory and stimulatorypathways mostly initiated by ligand-receptor interaction hardwiring theimmune system, specifically T-cell mediated immunity, to maintainself-tolerance and modulate the duration and amplitude of physiologicalresponses in peripheral tissues in order to minimize collateral tissuedamages normally (Pardoll, 2012). Tumor cells co-opt certain checkpointpathways as a major mechanism of immune resistance. For example,programmed cell death protein 1 ligand, PD-L1, is commonly up-regulatedon tumor cell surface of human cancers. The interaction of PD-L1 withits receptor, PD-1, expressed on tumor infiltrated lymphocytes (TILs),specifically on T cells, inhibits local T cell-mediated response toescape the immune surveillance (Liang et al., 2006; Sznol and Chen,2013). Thus, the inhibition of immunosuppressive signals on cancercells, or direct agonistic stimulation of T cells, results in and/orinduces a strong sustained anti-tumor immune response. Recent clinicalstudies strongly suggested blockage of immune checkpoint proteins viaantibody or modulated by soluble ligands or receptors are the mostpromising approaches to activating therapeutic antitumor immunity(Topalian et al., 2014). Currently, anti-PD-1 and anti-CTLA-4 (cytotoxicT-lymphocyte-associated antigen-4) antibodies have been approved by FDAto treat diseases such as melanomas.

Another co-stimulator molecule is the OX40 receptor (CD134), a member ofthe TNFR superfamily, which is membrane-bound and is expressed primarilyon activated CD4+ T cells (Paterson et al., 1987). Signaling through theOX40 receptor (hereinafter “OX40”) is costimulatory to effector T cellsand causes proliferation of T-cells (Watts, 2005; Weinberg et al.,1994). Studies of OX40 suggest that its major role is to dictate thenumber of effector T-cells that accumulate in primary immune responses,and consequently to govern the number of memory T-cells thatsubsequently develop and survive (Croft, 2003). A number in vitrostudies have been shown that OX40 provides a costimulatory signalresulting, in enhanced T cell proliferation and cytokine production.

Bi-Specific/Bi-Functional Antibody

The idea of using bispecific antibodies to efficiently retarget effectorimmune cells toward tumor cells emerged in the 1980s (Karpovsky et al.,1984; Perez et al., 1985; Staerz et al., 1985). Bispecific scaffolds aregenerally classified in two major groups with different pharmacokineticproperties, based on the absence or presence of an Fc fragment, IgG-likemolecules and small recombinant bispecific formats, most of themderiving from single chain variable fragment (scFv). Through theircompact size, antibody fragments usually penetrate tumors moreefficiently than IgG-like molecules but this benefit is mitigated by ashort serum half-life (few hours) limiting their overall tumor uptakeand residence time (Goldenberg et al., 2007). By contrast, the presenceof an Fc fragment, which binds to the neonatal Fc receptors, provides along serum half-life (>10 days) to the IgG-like formats, favoring tumoruptake and retention, but limits tumor penetration.

Recent studies have highlighted the therapeutic efficacy ofimmunotherapy, a class of cancer treatments that utilize the patient’sown immune system to destroy cancerous cells. Within a tumor thepresence of a family of negative regulatory molecules, collectivelyknown as “checkpoint inhibitors,” can inhibit T cell function tosuppress anti-tumor immunity. Checkpoint inhibitors, such as CTLA-4 andPD-1, attenuate T cell proliferation and cytokine production. Targetedblockade of CTLA-4 or PD-1 with antagonist monoclonal antibodies (mAbs)releases the “brakes” on T cells to boost anti-tumor immunity.Generating optimal “killer” CD8 T cell responses also requires T cellreceptor activation plus co-stimulation, which can be provided throughligation of tumor necrosis factor receptor family members, includingOX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest astreatment with an activating (agonist) anti-OX40 mAb augments T celldifferentiation and cytolytic function leading to enhanced anti-tumorimmunity against a variety of tumors. When used as single agents, thesedrugs can induce potent clinical and immunologic responses in patientswith metastatic disease (Linch et al., 2015).

SUMMARY

The present disclosure designed to investigate the bispecific antibodywith immunomodulatory aiming for the treatment of patient with cancers,such as prostate cancer, lung cancer, NSCLC, melanoma, lymphoma, breastcancer, head and neck cancer, RCC, or ovarian cancer were examined.

The present disclosure provides an antibody or an antigen-bindingportion thereof binding to OX40 (CD134), comprising: a heavy chainvariable region comprising an amino acid sequence of at least about 80%sequence homology to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 8, amino acid 128-246 of SEQ IDNO: 10, and amino acid 124-241 SEQ ID NO: 13; and a light chain variableregion comprising an amino acid sequence of at least about 80% homologyto the amino acid sequence selected from the group consisting of aminoacid 1-108 of SEQ ID NO: 5, 1-108 of SEQ ID NO: 7, 1-112 of SEQ ID NO:10, and 1-108 of SEQ ID NO: 13.

In one embodiment, the antibody or the antigen-binding portion thereofis a single chain variable fragment (scFv) sequence selected from thegroup consisting of SEQ ID NOs: 10, 11, 12, and 13.

In one embodiment, the antibody or the antigen-binding portion thereofis a bispecific antibody.

In one embodiment, the bispecific antibody comprises an immunecheckpoint protein binding site.

In one embodiment, the immune checkpoint protein binding site comprisesa programmed cell death protein 1 ligand (PD-L1) binding site, PD-1binding site, epidermal growth factor receptor (EGFR) binding site,human epidermal growth factor receptor 2 (HER2) binding site, cytotoxicT-lymphocyte-associated antigen 4 (CTLA-4) binding site, or lymphocyteactivation gene 3 (LAG3) binding site.

The present disclosure also provides an antibody or an antigen-bindingportion thereof binding to PD-L1, comprising: a heavy chain variabledomain comprising an amino acid sequence of at least about 80% sequencehomology to the amino acid sequence selected from the group consistingof SEQ ID NO: 2 and SEQ ID NO: 4; and a light chain variable domaincomprising an amino acid sequence of at least about 80% homology to theamino acid sequence selected from the group consisting of amino acid1-111 of SEQ ID NO: 1 and 1-110 of SEQ ID NO: 3.

The present disclosure also provides a bispecific antibody comprising atleast one of polypeptide chain, wherein the polypeptide chain comprisesan OX40 binding site and a PD-L1 binding site. The OX40 binding sitecomprises a heavy chain variable region comprising an amino acidsequence of at least about 80% sequence homology to the amino acidsequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO:8, amino acid 128-246 of SEQ ID NO: 10, and amino acid 124-241 SEQ IDNO: 13; and a light chain variable region comprising an amino acidsequence of at least about 80% homology to the amino acid sequenceselected from the group consisting of amino acid 1-108 of SEQ ID NO: 5,1-108 of SEQ ID NO: 7, 1-112 of SEQ ID NO: 10 and 1-108 of SEQ ID NO:13. The PD-L1 binding site comprises a heavy chain variable domaincomprising an amino acid sequence of at least about 80% sequencehomology to the amino acid sequence selected from the group consistingof SEQ ID NO: 2 and SEQ ID NO: 4; and a light chain variable domaincomprising an amino acid sequence of at least about 80% homology to theamino acid sequence selected from the group consisting of amino acid1-111 of SEQ ID NO: 1 and 1-110 of SEQ ID NO: 3.

In one embodiment, the polypeptide chain further comprises a Fc domain,a Fab fragment, and a scFv. The Fab fragment is connected to theN-terminus of the Fc domain, and the Fab fragment comprises the PD-L1binding site. The scFv is connected to the C-terminus of the Fc domain,and the scFv comprises the OX40 binding site.

In one embodiment, the polypeptide chain further comprises a linkerbetween the Fc domain and the scFv.

In one embodiment, the scFv comprises an amino acid sequence selectedfrom the group consisting of amino acid 455-707 of SEQ ID NO: 18,455-708 of SEQ ID NO: 19, 455-701 of SEQ ID NO: 20, 455-706 of SEQ IDNO: 21, 455-706 of SEQ ID NO: 22, 455-706 of SEQ ID NO: 23, 455-706 ofSEQ ID NO: 24, 455-706 of SEQ ID NO: 25, 455-706 of SEQ ID NO: 26,455-706 of SEQ ID NO: 27, 455-706 of SEQ ID NO: 28, and 455-706 of SEQID NO: 29.

In one embodiment, the bispecific antibody comprises one pairs ofpolypeptide chains.

In one embodiment, the bispecific antibody is an IgG, IgE, IgM, IgD,IgA, or IgY antibody.

In one embodiment, the bispecific antibody is an IgG antibody.

In one embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4antibody.

The present disclosure also provides an antibody-drug conjugatecomprising a therapeutic agent, and an antibody or an antigen-bindingportion binding PD-L1 and/or OX40, wherein the therapeutic agent iscovalently conjugated to the antibody or the antigen-binding portion bya linker.

In one embodiment, the antibody or an antigen-binding portion isselected from the above mentioned antibody or an antigen-bindingportion.

The present disclosure also provides a pharmaceutical compositioncomprising the antibody, the antigen-binding portion thereof, or thebispecific antibody as above mentioned, and at least onepharmaceutically acceptable carrier.

The present disclosure also provides a method of treating cancercomprising administering to the subject in need thereof an effectiveamount of the antibody, the antigen-binding portion thereof, or thebispecific antibody as above mentioned.

In one embodiment, the cancer is selected from the group consisting ofprostate cancer, lung cancer, Non-Small Cell Lung Cancer (NSCLC),melanoma, lymphoma, breast cancer, head and neck cancer, renal cellcarcinoma (RCC), and ovarian cancer.

The present disclosure also provides a nucleic acid molecule encodingthe antibody, the antigen-binding portion thereof, or the bispecificantibody as above mentioned.

It is to be understood that both the foregoing general description andthe following detailed description are by examples, and are intended toprovide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood by reading the followingdetailed description of the embodiment, with reference made to theaccompanying drawings as follows:

FIG. 1 shows immune checkpoints modulating T-cell mediated immunity.Antibody either agonistic or antagonistic against the checkpoints, suchas anti-ICOS, anti-CD28, anti-OX40, and anti-CD27, or anti-PD-1,anti-CTLA4, anti-LAG3, anti-BTLA, could be used to construct thebi-functional fusion protein depending on applications.

FIGS. 2A and 2B show the screening of phage clone by direct ELISA forPD-L1 expressed HEK293 cells.

FIGS. 3A and 3B show the screening of phage clone by cell-based ELISAwith OX40 expressed HEK293 cells.

FIG. 4 shows purified antibody leads specific for PD-L1 by SDS-PAGE withnon-reducing reagent to reveal the integrity and purity.

FIG. 5 shows purified antibody leads specific for OX40 by SDS-PAGE withnon-reducing or reducing reagent to reveal the integrity and purity.

FIG. 6 shows examples of the direct ligand binding activity of purifiedanti-immune check point proteins and anti-PD-L1 antibody leads againstPD-L1. Ligand pre-coated wells were first incubated with variousconcentrations of antibody leads as indicated. The bound proteins werethen detected with HRP conjugated goat anti-human IgG Fab specificantibody and OD₄₅₀ readings were plotted.

FIG. 7 shows examples of the direct ligand binding activity of purifiedanti-immune check point proteins and anti-OX40 antibody leads againstOX40. Ligand pre-coated wells were first incubated with variousconcentrations of antibody leads as indicated. The bound proteins werethen detected with HRP conjugated goat anti-human IgG Fab specificantibody and OD₄₅₀ readings were plotted.

FIG. 8 shows the flow analysis using PD-L1expression 293 cells. PD-L1expression HEK293 cells were first incubated with purified antibodyleads, and the bound antibodies were detected with Alexa-488 conjugatedgoat anti-human IgG (H+L) followed by fluorescence-activated cell sorter(FACS) analysis.

FIG. 9 shows the flow analysis using OX40 expression 293 cells. OX40expression HEK293 cells were first incubated with purified anti-OX40antibody leads, and the bound antibodies were detected with Alexa-488conjugated goat anti-human IgG (H+L) followed by FACS analysis. NS: nostaining.

FIG. 10 shows the blockage of PD-1/PD-L1 interaction with purifiedanti-PD-L1 antibodies. Purified antibodies as indicated were appliedwith biotinylated-PD-L1-Fc and recombinant human PD-1/His (hPD-1/His) toevaluate the inhibition activity of PD-1/PD-L1 interaction. The bindingrecombinant PD-L1-Fc and hPD-1/His was detected by streptavidin-HRP andanalysis by ELISA.

FIGS. 11A and 11B show anti-PD-L1 antibody leads with 1 or 10 µg/mLstimulates T-cell proliferation and induces IL-2 and/or IFN-γ productionin a mixed lymphocyte reaction (MLR) assay after 3 days (FIG. 11A) or 5days (FIG. 11B) antibody treatment.

FIG. 12A shows the ability of anti-OX40 antibody leads to enhance theCD3+ T cell activation with dosage response as well as referenceantibody. FIG. 12B shows the concentration of human IL-2 and IFN-γpresent in cell culture media following 3 days of stimulation of human Tcells with plate bound anti-CD3 and several concentrations of anti-OX40antibody leads.

FIGS. 13A and 13B show the concentration of human IL-2 (FIG. 13A) andIFN-γ (FIG. 13B) present in cell culture media following 3 days ofstimulation of human T cells with plate bound anti-CD3 and severalconcentrations of OX40 specific antibody leads.

FIG. 14 shows the structure of an antibody heavy chain Fc fused with anOX40 specific scFv domain.

FIG. 15 shows examples of PAGE-gel analysis of anti-immune check pointantibodies-human OX40 fusion proteins. Purified fusion proteins,anti-PD-L1-OX40 scFv fusion proteins were shown to have a molecularweight about 220 kDa (non-reducing), and heavy chain fusion has about 85kDa and light chain is about 25 kDa (reduced) in both antibody fusions.

FIGS. 16A and 16B show bispecific antibody synergic stimulates T-cellactivation for IL-2 and IFN-γ production in a mixed lymphocyte reaction(MLR) assay after 3 days (FIG. 16A) or 5 days (FIG. 16B) with mono-,combined or anti-PD-L1-OX40 scFv bispecific antibody treatment.

FIGS. 17A to 17E respectively show the aggregation and puritydetermination of Bi-specific antibodies, Anti-PD-L1-OX40 Ab andAnti-PD-L1-OX40 Ab-V1 to V4, with 5 different linkers in OX40 scFv.

FIG. 18 shows sequence variants among OX40 clone B17 scFv ofAnti-PD-L1-OX40 Ab-V4 to V12 (SEQ ID NO: 30-38).

FIG. 19 shows examples of PAGE-gel analysis of anti-immune check pointantibodies-human OX40 fusion proteins. Purified fusion proteins,anti-PD-L1-OX40 Ab-V5 fusion proteins were shown to have a molecularweight about 220 kDa (non-reducing), and heavy chain fusion has about 80kDa and light chain is about 30 kDa (reduced) in both antibody fusions.

FIG. 20 shows a flow chart illustrating the ELISA method for bindingactivity evaluation of bispecific antibody variants.

FIG. 21 shows the human PD-L1 binding activity of the bispecificantibody variants and its EC50.

FIG. 22 shows the human OX40 binding activity of the bispecific antibodyvariants and its EC50.

FIG. 23 shows the ex vivo serum stability of bispecific antibodyvariant, anti-PD-L1-OX40 Ab-V5.

FIGS. 24A and 24B show the IL-2 production for 3 days (FIG. 24A) andIFN-γ production for 5 days (FIG. 24B) after modulating T cell withmono-, combined or anti-PD-L1-OX40 Ab-V5 bispecific antibody treatment.

FIG. 25 is a graph showing the effect of anti-PD-L1-OX40 Ab-V5bispecificantibody treatment and monoclonal antibody treatment on the growth ofPC-3 tumor in Fox Chase SCID®Beige mice.

DETAILED DESCRIPTION

Reference will now be made in detail to the present embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers are used in thedrawings and the description to refer to the same or like parts.

The present invention describes the expression, purification andcharacterization of bi-functional proteins with isolated functionalagonistic OX40 scFv fused to the C-terminus of Fc domain of anti-immunecheckpoint protein antibodies. These proteins interact with itscorresponding check point target shall transmit the inhibitory orstimulatory signal to modulate T-cell involved immunity. The componentsof Fc fusion proteins in present invention are of all human origins, andthus are expected to be non-immunogenic and can be used as therapeuticsin human.

Bispecific molecules such as bispecific antibodies (BsAbs) provide ameans of simultaneously targeting multiple epitopes on the samemolecular target or different targets with a single therapeutic agent.As cancer therapeutics, they have the potential to confer novel or morepotent activities, lower the cost of goods and facilitate thedevelopment of new therapeutic regimens in contrast to a mixture of twomAbs (Chames and Baty, 2009; Hollander, 2009; Thakur and Lum, 2010).Recently, catumaxomab, a trifunctional bispecific antibody targetinghuman epithelial cell adhesion molecule (EpCAM) and CD3 has shown aclear clinical benefit in patients with peritoneal carcinomatosis ofepithelial cancers (Heiss et al., 2010), and a bispecific T-cellengaging (BiTE) antibody with dual specificity for CD19 and CD3 has alsodemonstrated encouraging clinical activity in patients with CD19expressing hematological malignancies (Bargou et al., 2008). Despitestrong interest in the development of bispecific molecules as cancertherapeutics, technical challenges in the production of stable andactive bispecific molecules have in the past hindered the clinicalevaluation of most bispecific formats. Many engineered antibody formats,including an IgG-like bispecific antibody have compromised stability orsolubility (Bargou et al., 2008; Demarest and Glaser, 2008; Lu et al.,2005). Furthermore, several strategies have been taken to increase theproduct quality and in vivo stability of bispecific molecules, includingPEGylation, conjugation with human serum albumin and Fc engineering(Muller et al., 2007; Ridgway et al., 1996). Bispecific single chainantibodies of the general form described above have the advantage thatthe nucleotide sequence encoding the four V-domains, two linkers and onespacer can be incorporated into a suitable host expression organismunder the control of a single promoter. This increases the flexibilitywith which these constructs can be designed as well as the degree ofexperimenter control during their production. In addition, the Fc of IgGis a very another attractive scaffold for designing novel therapeuticsbecause it contains all antibody functions except the binding ability.Fc engineering is important for improving the effectiveness of thebispecific antibodies. Therefore, the IgG-based conformation is using inpresent invention for two independent target on immune cells or targetcell in immunotherapy.

Targeting immune-check point proteins are promising approaches toactivate antitumor immunity. Anti-check point proteins, such as PD-1,PD-L1, CTLA-4, LAG3, etc., are currently evaluated clinically (FIG. 1 ).Preliminary data with blockers of immune checkpoint proteins have beenshown to be able to enhance antitumor immunity with the potential toproduce durable clinical responses. However, despite the remarkableclinical efficacy of these agents in a number of malignancies, it hasbecome clear that they are not sufficiently active for many patients.Numerous additional immunomodulatory pathways as well as inhibitoryfactors expressed or secreted by myeloid and stromal cells in the tumormicroenvironment are potential targets for synergizing with immunecheckpoint blockade. Therefore, combining anticancer or bispecificantibody therapies has been essential to achieve complete remission andcures for patients with cancer.

The present invention describes the construction, expression andcharacterization of anti-immune checkpoint protein antibody Fc fusedwith different immune checkpoint protein specific scFv protein. TheC-terminally positioned OX40 scFv in fusion constructs shall allowexpanding the power of fusion proteins beyond OX40 activation approachif the fusion counterpart is immune system potentiating agent, such asanti-EGFR, anti-HER2, or anti-CTLA-4 antibody, for example.

Antibody Generation From OmniMab Library

For the generation of therapeutic antibodies against PD-L1 or OX40,selections with the OmniMab phagemid library were carried out. Thephagemid library is generated by AP Biosciences Inc. (APBio Inc.) from acollection of over hundred health donors B cells. Phages for the 1stround of pannings were prepared by Hyperphage (M13K07ΔρIII, Progen,Heidelberg, Germany). Solid phase panning and cell panning against PD-L1or OX40 were applied for PD-L1 or OX40 specific binder selection andisolation from OmniMab library. Solid phase panning was performed usingrecombinant human PD-L1-Fc or OX40-Fc (APBio Inc.) in the first roundselection and then HEK293 cells expressed PD-L1 or OX40 were used fortwo and three round enrichment. After three rounds selection, thespecific PD-L1 or OX40 binders were screened and isolated by directELISA or cell-based ELISA with corresponding recombinant protein (FIG.2A 2B, 3A, and 3B). Pre-coated PD-L1-Fc recombinant proteins or OX40expressed 293 cells were blotted with supernatant containing rescuedphages for 1 hour and washed with PBS containing 0.1% Tween-20 for threetimes. Bound phages were detected by HRP conjugated anti-M13 antibody(Roche) and TMB substrate was used for signal development. The OD450readings were recorded. The positive binders were isolated and sent forsequencing to confirm the sequence and diversity of heavy chain andlight chain. The variable region of heavy chain and light chain specificto PD-L1 or OX40 were described from the SEQ ID NO: 1 to SEQ ID NO: 8:SEQ ID NO: 1 is the light chain of PD-L1 clone 6, SEQ ID NO: 2 is thevariable region of heavy chain of PD-L1 clone 6, SEQ ID NO: 3 is thelight chain of PD-L1 clone 32, SEQ ID NO: 4 is the variable region ofheavy chain of PD-L1 clone 32, SEQ ID NO: 5 is the light chain of OX40clone B17, SEQ ID NO: 6 is the variable region of heavy chain of OX40clone B17, SEQ ID NO: 7 is the light chain of OX40 clone B19, SEQ ID NO:8 is the variable region of heavy chain of OX40 clone B19. As shown inthe FIGS. 2A, 2B, 3A and 3B, several clones were isolated and known tobe recognized specifically for corresponding antigen as comparing withnegative control.

Subcloning and Expression/Purification of Selected PD-L1 or OX40Specific Binder as IgG Format

To facilitate the quick screening of specific binder with functionalityin T cell activation, the heavy chains and light chains of positivebinders against PD-L1 or OX40 by ELISA were then amplified, digested andsub-clone into APBio specialized IgG expression vector carrying IgG4constant region (SEQ ID NO: 9). After sequence validation, the plasmidswere then prepared and transfected into HEK293 cells for antibodyexpression with 293 fectin transfection reagent (Invitrogen). After 4days culture, the antibody secreted into serum-free medium is affinitypurified from culture supernatant by Protein G chromatography. Purifiedantibody is then concentrated, followed by dialysis in PBS buffer. Thefinal concentration of dialyzed protein is determined by NanoDrop2000spectrophotometer and the purity and integrity are determined bySDS-PAGE with or without reducing reagent as shown in the FIGS. 4 and 5. The integrity of various purified antibody leads, either PD-L1specific or OX40 specific, is normal in the HEK293 cells as well asreference antibody, MPDL3280A for PD-L1 or GSK3174998 for OX40.

In one embodiment, the present disclosure provides an antibody or anantigen-binding portion thereof binding to OX40 (CD134), comprising aheavy chain variable region and a light chain variable region. The heavychain variable region comprises an amino acid sequence of at least about80% sequence homology to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 8, amino acid 128-246 of SEQ IDNO: 10, and amino acid 124-241 SEQ ID NO: 13. In some examples, theheavy chain variable region comprises an amino acid sequence of at leastabout 85%, 90%, or 95% sequence homology to the amino acid sequence asabove mentioned. The light chain variable region comprising an aminoacid sequence of at least about 80% homology to the amino acid sequenceselected from the group consisting of amino acid 1-108 of SEQ ID NO: 5,1-108 of SEQ ID NO: 7, 1-112 of SEQ ID NO: 10, and 1-108 of SEQ ID NO:13. In some examples, the light chain variable region comprises an aminoacid sequence of at least about 85%, 90%, or 95% sequence homology tothe amino acid sequence as above mentioned.

In one embodiment, the present disclosure provides an antibody or anantigen-binding portion thereof binding to PD-L1, comprising a heavychain variable domain and a light chain variable domain. The heavy chainvariable domain comprises an amino acid sequence of at least about 80%sequence homology to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 2 and SEQ ID NO: 4. In some examples, the heavychain variable region comprises an amino acid sequence of at least about85%, 90%, or 95% sequence homology to the amino acid sequence as abovementioned. The light chain variable domain comprises an amino acidsequence of at least about 80% homology to the amino acid sequenceselected from the group consisting of amino acid 1-111 of SEQ ID NO: 1and 1-110 of SEQ ID NO: 3. In some examples, the light chain variableregion comprises an amino acid sequence of at least about 85%, 90%, or95% sequence homology to the amino acid sequence as above mentioned.

Binding Activity Determination for PD-L1, OX40 Specific IgG Leads byDirect ELISA

Purified antibody leads against PD-L1 or OX40 (anti-PD-L1 antibody leadsor anti-OX40 antibody leads) were then applied for ELISA bindingcharacterization on human PD-L1-Fc or OX40-Fc in a direct coated setup.FIGS. 6 and 7 showed the ELISA binding result for anti-PD-L1 andanti-OX40 antibodies, respectively. For PD-L1 specific antibodies, mostleads showed a similar or better binding activity with referenceantibody (Ref Ab, MPDL3280A, Roche).

Purified human PD-L1 or OX40 IgG1 Fc chimera (PD-L1-Fc or OX40-Fc,APBio) was dialyzed in Phosphate Buffered Saline (PBS), adjusted to 1mg/mL and then diluted with PBS to a final concentration of 1 µg/mL.Nunc-Immuno Maxisorp 96 well plates were coated with 0.1 mL per well ofrecombinant PD-L1-Fc or OX40-Fc chimera leaving empty wells fornonspecific binding controls and incubated at 4° C. overnight. ThePD-L1-Fc or OX40-Fc chimera solution was removed and the plates werewashed three times with 0.4 mL wash buffer (0.1 % Tween-20 in PBS). 0.4mL blocking buffer (5% low-fat milk powder in PBS) was added to allwells and incubated at room temperature for 1 hour with mixing. Theblocking buffer was removed and plates washed three times with 0.4 mLwash buffer. Serial dilutions of the PD-L1 or OX40 test antibodies wereprepared in PBS and 0.1 mL diluted Ab was added per well. Plates wereincubated 1 hour at room temperature. Antibody solution was removed andthe plates washed three time with 0.4 mL wash buffer per well.Horseradish peroxidase labeled goat anti-human IgG, F(ab′)₂ specificF(ab′)₂ antibody (Jackson Immunoresearch #109-036-097) was diluted1:2000 with PBS and added 0.1 mL per well. The plates were incubated 1hour at room temperature and washed with 0.4 mL per well wash buffer.0.1 mL TMB reagent (Invitrogen) was added and incubated for 1 to 5minutes at room temperature. The reaction was stopped by adding 0.05 mL1 N HCl and absorbance was read at 450 nm on a Bio-Tek Spectra.Calculated EC50 for anti-PD-L1 antibody leads to PD-L1 showed most leadspossess good binding activity as well as MPDL3280A (Ref Ab) by directELISA (FIG. 6 ). On the contrary, most anti-OX40 antibody leads showedmuch lower binding activity as comparing with reference antibody (RefAb, GSK3174998)(FIG. 7 ).

Binding Activity Determination for PD-L1 and OX40 Specific IgG Leads byFACS

Purified antibody leads (anti-PD-L1 antibody leads or anti-OX40 antibodyleads) were also applied for flow cytometry to determine and compare thebinding activity with PD-L1 or OX40 expressed HEK293 cells. FIGS. 8 and9 show the binding activity of corresponding antibody leads as indicatedby FACS with stable expressed PD-L1 or OX40 HEK293 cells.

FACS analysis of PD-L1 stable expression 293 cells stained withanti-PD-L1 antibody leads to examine the PD-L1 binding activity, stableexpression cells were incubated with 1 µg/mL purified anti-PD-L1antibody leads, reference antibody (Ref Ab MPDL3280A) or with isotypeantibody as negative control on ice for 1 hr. The cells were washedthree times with 1× PBS and then incubated with Alexa-488-conjugatedgoat anti-human IgG (H+L) (Invitrogen Inc.) on ice for additional 1 hr.After staining, the cells were washed three times with 1× PBS,resuspended in 1× PBS/2%FBS before analyzed by FACS Calibur (BDBiosciences, Inc.) and FlowJo (TreeStar, LLC). Same scenario, thebinding activity of anti-OX40 antibody leads for stable expressed OX40HEK293 cells in FIG. 9 were also executed with a similar strategy andanalyzed as described above. As shown in the FIG. 8 , most anti-PD-L1antibody leads possess a good binding activity as well as referenceantibody. This indicated the phage clones selected from the OmniMablibrary indeed recognize the native PD-L1 in the cells.

This phenomenon is also observed for anti-OX40 antibody leads as shownin the FIG. 9 . FACS analysis of OX40 stable expression 293 cells clone2D5 stained with purified anti-OX40 antibodies leads to examine the OX40binding activity, stable expression cells were incubated with 2 µg/mLanti-OX40 reference Abs (OX40 ref.) or anti-CD137 reference Abs (CD137ref.) as control antibody on ice for 1 hr. The cells were washed threetimes with 1× PBS and then incubated with Alexa-488-conjugated goatanti-human IgG (H+L) (Invitrogen Inc.) on ice for additional 1 hr. Afterstaining, the cells were washed three times with 1× PBS, resuspended in1× PBS/2%FBS before analyzed by FACS Calibur (BD Biosciences, Inc.) andFlowJo (TreeStar, LLC).

Ligand Competition Binding (ELISA Assay)

Antibody leads were exposed to the binding selectivity and affinityassay used to evaluate the anti-PD-L1 antibody leads of the presentinvention for their ability to block binding of PD-L1 to PD-1.

Antibodies were tested for their ability to block the binding of thehuman PD-L1-Fc chimera (PD-L1-Fc) to recombinant human PD-1/His(hPD-1/His) by ELISA. Purified recombinant hPD-1/His (APBio) wasdialyzed to 1 mg/mL in PBS and then conjugated with biotin (Abcam). NuncMaxisorp 96 well pate was coated with 250 ng hPD-1/His per well in PBSovernight. The hPD-1/His solution was removed and the plates were washedthree times with 0.4 mL wash buffer (0.1 % Tween-20 in PBS). 0.4 mLblocking buffer (5% low-fat milk powder in PBS) was added to all wellsand incubated at room temperature for 1 hour with mixing. During theblocking step the antibody stocks were diluted in a range from 200 nM to0 nM in PBS with 2 folds serial dilution. Purified recombinantbiotinylated-PD-L1-Fc chimera was diluted to 4 µg/mL in PBS. ThePD-1/His coated plates were washed three times with 0.2 mL wash buffer(0.1 % Tween 20 in PBS). 60 µL antibody dilutions (anti-PD-L1 antibodyleads or Ref Ab MPDL3280A) were added alone with 60 µLbiotinylated-PD-L1-Fc chimera and incubated at room temperature for 1hour. Plates were washed as described previously. Streptavidin-HRP wasdiluted 1:2000 in PBS, 100 µL of the resulting solution added to thewells of the washed plated, and incubated at room temperature for 1hour. Plates were washed as previously described, 100 µL TMB substratesolution was added to each well and incubated for 10 minutes. Thereaction was stopped with 50 µL 1 N HCl and absorbance at 450 nm readusing Bio-Tek reader and shown in FIG. 10 . Partial antibody leads areshown to inhibit the interaction between PD-PD-L1 by competition ELISA.Most antibody leads revealed a similar blocking activity as comparingwith reference antibody (Ref Ab MPDL3280A).

Enhanced Stimulation of T Cell Activation by Inhibition of PD-1:PD-L1Ligand Interaction for Anti-PD-L1 Antibody

The PD-1 signaling pathway inhibits moderate TCR/CD28 costimulatorysignals, with cytokine production being reduced first without a decreasein T cell proliferation. As the TCR/CD28 costimulatory signals weaken,the PD-1 pathway dominates, with a great reduction in cytokineproduction accompanied by a reduction in proliferation. Accordingly inorder to confirm that the inhibition of the PD-1 via inhibition of theinteraction with PD-L1, human antibodies of the invention enhances Tcell activation, mixed lymphocyte reactions (MLRs) are performed.

Monocytes from human whole blood were enriched by RosetteSep™ HumanMonocyte Enrichment Cocktail (Cat. No.15068) and cultured indifferentiation medium, RPMI 1640 with 10%FBS, 100 ng/mL (1000 U/mL)GM-CSF, 100 ng/mL (500 U/mL) for 6 days. The differentiate dendriticcells (DC) from monocyte were checked by DC-SIGN-PE, anti-CD14conjugated with FITC Ab, anti-CD83 conjugated with PE Ab, or anti-CD80conjugated with FITC Ab to validate the differentiation and used to beAPCs in MLRs.

Allogenic CD4+ T cells from human whole blood were isolated byRosetteSep™ Human CD4+ T Cell Enrichment Cocktail (Cat. No. 15062). Thepurity of CD4+ T cells were checked with anti-CD4 conjugated APC Ab tomake sure the purity is above 95% and then labeled with 1 uM CFSE(CellTrace™ CFSE cell proliferation kit, Life technologies, Cat. No.C34554) for T cells proliferation assay. Labeled CD4+ T cells were usedto co-culture with immature DC with different antibody leads asindicated for 3 and 5 days to see whether the antibody leads couldrestore the T cell activation through blocking the interaction betweenPD-1 and PD-L1. After 3 and 5 days incubation, the supernatant werecollected for cytokine, such as IL-2 and IFN-γ quantitation by ELISA.The addition of anti-PD-L1 antibody leads (clones 6, 32, 28, 51, 64, 27,and 37) to cultures of immature dendritic cells plus allogeneic T cellsis predicted to result in an increase in T cell proliferation andcytokine production, as compared to isotype IgG (iso#1, #2) treatedcultures and shown in the FIGS. 11A and 11B. The IL-2 and IFN-γproduction increase significantly in the MLRs as comparing with isotypeantibody treatment after 3 days (FIG. 11A) or 5 days (FIG. 11B) antibodytreatment, especially for anti-PD-L1 antibody clone 6. The cytokineincrement is still obviously after 5 days antibody treatment and similarto reference antibody (ref), MPDL3280A. This indicated the anti-PD-L1antibody clone 6 should be one of the potential leads for bispecificantibody composite.

Agonistic Activity Assay of Anti-OX40 Antibody

In order to activate OX40 costimulation of T-cell proliferation andcytokine production, the purified antibody leads were functionallyscreened for their ability to enhance cytokine production,proliferation, and to induce proliferation in human CD3+ T-cells. Theanti-CD3 antibody (OKT3, BioLegend Cat. No.317304) and anti-OX40antibody leads (clones B6, B70, B120, A4, B17, B19, and B30), referenceantibody (GSK3174998) or isotype antibodies (iso#1, #2) were coated inthe Maxisorp 96-well plate. Meanwhile, naïve human CD3+T-cells wereisolated from the human blood from heathy adult volunteers using acommercially available RosetteSep™ Human T Cell Enrichment Cocktail(STEMCELL Cat. No.15061) as manufacture’s described. The isolated CD3+ Tcells were then labeled by CFSE (CellTrace™ CFSE cell proliferation kit,Life technologies, Cat. No. C34554) and seeded as 1 × 10⁶ cells/mL intothe antibody pre-coated well containing RPMI 1640 medium, 10% fetalbovine serum and 2.5 mM L-glutamine to determine the cell proliferationand cytokine production. After 3 days culture, the cells were collectedfor proliferation assay by flow cytometry and medium were then analyzedfor IL-2 and IFN-γ production by quantitation ELISA.

The screening of anti-OX40 antibody leads with agonistic activity in Tcell activation was shown in the FIG. 12A. All anti-OX40 antibody leadsshowed the ability to enhance the CD3+ T cell activation with dosageresponse as well as reference antibody. Higher dosage antibody treatmentshowed obviously higher T cell activation activity. Meanwhile, cytokineproduction (FIG. 12B), such as IL-2 and IFN-γ also revealed similar Tcell activation response, especially for anti-OX40 antibody lead cloneB17. Cytokine is highly inducted after anti-OX40 antibody lead B17 3days treatment. The enhancement is much higher than reference antibodytreatment, this implicated clone B17 should be one of the candidates forbispecific antibody construction.

As the data shown in the FIGS. 13A and 13B, both anti-OX40 antibodyleads, clones B17 and B19, were shown a better agonistic activity in theassay after anti-OX40 antibody leads (B17 or B19) 3 days treatment.Either IL-2 production or IFN-γ production shows an obvious enhancementupon antibody treatment and revealed does-dependent correlation. Highercytokine productions were recorded in higher dose antibody treatment.

In order to evaluate the agonistic activity of OX40 antibody leads, B17and B19, the EC50 were also determined as well as agonistic activityassay and cytokine production were recorded for comparison.

Construction, Expression and Purification of Anti-PD-L1-OX40 scFvAntibody

Since the bispecific is designed as IgG based fused with scFv format,the structure of anti-immune checkpoint antibody Fc-terminally fusedwith OX40 scFv. Antibody can be inhibitory anti-immune checkpointantibodies, such as anti-PD-L1, anti-PD-1, anti-CTLA4, anti-LAG3, etc.,or stimulatory antibodies, such as anti-CD28, anti-CD137, anti-CD27,anti-ICOS, etc. A linker is placed between antibody Fc and OX40 scFv togenerate the bispecific antibody as depicted in FIG. 14 .

In some embodiment, the anti-PD-L1 antibody lead clone 6 is assigned tobe IgG form, on the other hand, the anti-OX40 antibody lead would betransformed as scFv format to fuse at C-terminus of Fc region inanti-PD-L1 antibody lead clone 6. The transformation from antibody toscFv format could result in the reduction of the binding activity orspecificity; therefore several anti-OX40 antibody leads were used toscFv transformation. Construction of bi-functional anti-PD-L1 antibodyFc fused with full-length OX40 scFv (SEQ ID NO: 10 as clone A4, SEQ IDNO: 11 as clone B17, SEQ ID NO: 12 as clone B19, or SEQ ID NO: 13 asclone B120). A short flexible peptide linker, (GGGGS)₂ (SEQ ID NO: 14)was placed between, for example, anti-PD-L1 antibody heavy chainC-terminus of Fc region and N-terminal module of OX40 scFv to ensurecorrect folding and minimize steric hindrance. The coding sequences ofanti-PD-L1-OX40 scFv antibodies were shown in SEQ ID NO: 16(anti-PD-L1-clone 6 heavy chain-OX40 clone B17 scFv) and NO: 17(anti-PD-L1-clone 6 heavy chain-OX40 clone B19 scFv). The constructedantibody Fc fusion proteins were leaded by a signal peptide (SEQ ID NO:15) and expressed by mammalian cells, and purified from the transfectedcell culture supernatant via 1-step Protein G chromatography. As shownin FIG. 15 , greater than 90% purity can be obtained in a single steppurification process and shows that purified fusion proteins havecorrect molecular weight (Mw = 220 kD).

Enhanced Stimulation of T Cell Activation for Anti-PD-L1-OX40 scFvBispecific Antibody Leads in MLRs

To determine the synergic cooperation of bispecific antibody inenhancing T cells activation through inhibition the interaction betweenPD-1 and PD-L1 and agonistic activation of OX40 signaling, thebispecific antibody leads, anti-PD-L1-OX40 scFv, were applied into MLRsas described above. IL-2 and IFN-γ production were then recorded after 3or 5 days antibody treatment. Mono-, combination or bispecific antibodywas applied as equal amount or equal mole to compare the synergic effectin T cell activation enhancement and isotype IgG was used a negativecontrol. As the data shown in the FIGS. 16A and 16B, the anti-PD-L1antibody leads alone showed a significant IL-2 induction after 3 daystreatment as well as reference antibody, MPDL3280A, on the contrary, theanti-OX40 antibody leads is unable to increase obviously upregulation ofcytokine production, either after 3 days or 5 days antibody treatment.This is consisted with reference antibody, GSK3174998. However,combination of the anti-OX40 antibodies and anti-PD-L1 antibodies showeda significant upregulation of cytokine production after 3 and 5 daysantibody treatment. The synergic effect is also observed in thebispecific antibody leads treatment and increment of cytokine productionis similar as well as combination treatment. This indicated theanti-PD-L1-OX40 scFv bispecific antibody leads also function as well asantibody combination treatment without loss any binding activities inthe scFv transformation.

Aggregation and Purity Determination of Bi-Specific Antibody

Since purified anti-PD-L1-clone 6-OX40 clone B17 scFv Ab revealed alower purity (74.07%) by SEC-HPLC analysis after a single column proteinA chromatography purification, therefore, several antibody variants weregenerated to improve the purity and reduce the aggregation for thebispecific antibody in the present invention. The linkers described asabove were used to replace the linker in OX40 B17 scFv in the bispecificantibody, anti-PD-L1-OX40 Ab (SEQ ID NO: 16), and produced asanti-PD-L1-OX40 Ab-V1 to V4 (SEQ ID NO: 18 to SEQ ID NO: 21) in the CHOcells. Those variants were then purified and analyzed by XBridge ProteinBEH SEC-HPLC column (Waters, Cat. No.186007640). The data was summarizedas below Table 1, one of the bispecific antibody variants,anti-PD-L1-OX40 Ab-V4 revealed a significant improvement of antibodypurity. The purity is enhanced from 74.07 to 92.27%. Therefore, theanti-PD-L1-OX40 Ab-V4 was used to engineer further to improve theantibody purity.

TABLE 1 Different linkers in OX40 B17 scFv Abbreviation Heavychain/light chain Linker in OX40 B17 scFv Reference Anti-PD-L1-OX40 AbAnti-PD-L1-6-OX40 B17 scFv-L1 HC/ Anti-PD-L1 6 LC GGGGSGGGGSGGGGS (SEQID NO: 39) Int. J. Mol. Sci. 2014, 15(12), 23658-23671 Anti-PD-L1-OX40Ab-V1 Anti-PD-L1-6-OX40 B17 scFv-L2 HC/ Anti-PD-L1 6 LC SSGGGGSGGGGGGSSRSSL (SEQ ID NO: 40) None Anti-PD-L1-OX40 Ab-V2 Anti-PD-L1-6-OX40 B17scFv-L3 HC/ Anti-PD-L1 6 LC GGKGSGGKGTGGKGS GGKGS (SEQ ID NO: 41) VirolJ. 2008; 5:21 Anti-PD-L1-OX40 Ab-V3 Anti-PD-L1-6-OX40 B17 scFv-L4 HC/Anti-PD-L1 6 LC GSASAPTLFPLVS (SEQ ID NO: 42) DOI: 10.3892/mmr.2013.1502Anti-PD-L1-OX40 Ab-V4 Anti-PD-L1-6-OX40 B17 scFv-L5 HC/ Anti-PD-L1 6 LCGSTSGSGKPGSGEGS TKG (SEQ ID NO: 43) PMID: 8309948

For characterization the size distribution of bi-specific antibodies,samples were loaded onto XBridge Protein BEH SEC-HPLC column (Waters,Cat. No.186007640) using a Waters Alliance 2695 Separations Module.Protein peak were detected at 280 nm using a Water 2996 PDA Detector.The mobile phase was isocratic 25 mM sodium phosphate (Sigma, Cat.No.04272 and Cat. No.04269) with 200 mM NaCl (AMRESCO, Cat. No.0241), pH6.8, at a flow rate of 0.4 mL/min. Peak percentages were determined bythe portions of peak area as shown in FIGS. 17A to 17E.

Anti-PD-L1-OX40 Ab-V4 revealed a significant purity improvement (FIG.17E). The bispecific antibody was engineered further in the OX40 B17scFv fragment to improve purity again. Several residues in the OX40 B17scFv showed in FIG. 18 were substituted with difference amino acid andheavy chain variants were pairing with anti-PD-L1 clone 6 light chain togenerate several bispecific antibody variants, from anti-PD-L1-OX40Ab-V5 to V12 (SEQ ID NO: 22 to SEQ ID NO: 29), and then expressed andpurified as mentioned above. The purity of bispecific antibody variantswere summarized as below Table 2, the anti-PD-L1-OX40 scFv-V5 revealedthe best purity in those antibody variants. The purity is aroused up to96.46%. This is shown a superior purity for the engineered bispecificantibody and also revealed a good development ability for thisbispecific antibody in the future. As shown in FIG. 19 , the integrityof anti-PD-L1-OX40 Ab-V5 was also analyzed by SDS-PAGE and shown a goodintegrity under reducing and non-reducing condition.

TABLE 2 Purity of Antibody Antibody Purity by SEC-HPLC (%)Anti-PD-L1-OX40 Ab-V4 92.27 Anti-PD-L1-OX40 Ab-V5 96.46 Anti-PD-L1-OX40Ab-V6 86.36 Anti-PD-L1-OX40 Ab-V7 88.04 Anti-PD-L1-OX40 Ab-V8 90.00Anti-PD-L1-OX40 Ab-V9 87.89 Anti-PD-L1-OX40 Ab-V10 86.56 Anti-PD-L1-OX40Ab-V11 86.61 Anti-PD-L1-OX40 Ab-V12 84.78

Meanwhile, the engineered bispecific antibody variants were also appliedfor binding activity evaluation for human PD-L1 and OX40 by direct ELISAas shown in FIG. 20 . All bispecific antibody variants as indicatedshowed the same binding activity for human PD-L1 (FIG. 21 ), thisbinding activity is similar with anti-PD-L1 6 antibody. This phenomenonwas also observed in the human OX40 binding assay (FIG. 22 ). Onlyanti-PD-L1-OX40 Ab-V10 showed a weaker binding activity for human OX40as comparing with other variants. It indicated the engineering of OX40scFv is not affected the OX40 binding activity. The binding activity isretained either for PD-L1 or OX40. Since the anti-PD-L1-OX40 Ab-V5revealed a superior antibody purity and binding activity for PD-L1 andOX40, so the anti-PD-L1-OX40 Ab-V5 was chosen for serum stability.

Ex Vivo Serum Stability

The stability was assessed in human serum (BioreclamationIVT, Cat.No.HMSRM) as well as serum from relevant preclinical species: rhesusmonkey (BioreclamationIVT, Cat. No.RHSSRM), and CD1 mouse(BioreclamationIVT, Cat. No.MSESRM). Samples were added into differentspecies serum for a final concentration of 15 µg/mL and incubated at 37°C. water bath. Serum samples were collected after incubation times of 0,1, 2, 3, 7, 10 and 14 day and stored frozen at -80° C. until analysis.

Quantitation Sandwich ELISA

OX40-Fc was coated into ELISA plate (NUNC, Cat. No.442404) with 100 µLat 1 µg/mL in PBS and incubated for overnight at 4° C. Wash buffer wasprepared as PBS with 0.1% Tween-20 (Sigma, Cat. No.P2287-500 mL) andblocking buffer was prepared as 1% BSA (UniRegion, Cat.No.UR-BSA001-100G) in wash buffer. Serum samples were prepared with10-fold dilution with 3x serial dilution in blocking buffer and thestandards were prepared at 10 nM with 3x serial dilution in blockingbuffer. Biotinylated PD-L1-Fc was labeled with Biotin Fast conjugationKit (abcam, Cat. No.ab201796) using standard protocol and prepared at 30nM in blocking buffer. Streptavidin-HRP (abcam, Cat. No.ab7403) wasprepared at 1 µg/mL in blocking buffer. All the samples were added intoeach well for 100 µL after plates washed 3 times with wash buffer andincubated for 1 hour at ambient temperature. TMB development with 100 µLTMB solution (Invitrogen, Cat. No.00-2023) for 2 min and stopped with100 µL 1 N HCl solution (Merck, Cat. No.1.00317.1000). O.D. 450 nmabsorption was read by ELISA reader (Biotek, Powerwave XS).

Anti-PD-L1-OX40 Ab-V5 was chosen for ex vivo serum stability because ofits superior purity and binding activity for PD-L1 and OX40. Thepurified bispecific antibodies were mixed with serum from differentspecies, such as human, mouse or monkey. After several days culture, thesamples were took and analyzed by sandwich ELISA to determine theantibody amount. As shown in FIG. 23 , the anti-PD-L1-OX40 Ab-V5 stillshowed a good serum stability after 14 days culture at 37° C. Theconcentration of the antibody is still above 70% either in human, mouseor monkey. It is indicated the antibody also have a good serumstability.

To measure the ability of the anti-PD-L1-OX40 Ab-V5 to modulate T cellresponsiveness purified T cells will be cultured with allogeneicdendritic cells, prepared by culturing monocytes in GM-CSF and IL-4 forfew days. Parallel plates were set up to allow collection ofsupernatants at day 3 and day 5 to measure IL-2 and IFN-γ respectivelyusing a commercial ELISA kit. As the data shown in FIGS. 24A and 24B,the IL-2 and IFN-γ production are highly upregulated in the bispecificantibody treatment (V5) as well as combination treatment after 3 or 5days antibody treatment. Also, the enhancement is obviously superiorthan the anti-PD-L1 Ab or anti-OX40 Ab treatment alone. This implicatedthe engineered bispecific antibody, V5, still possess the agonisticactivity as well as combination treatment without functionality lost andcould be developed as a therapeutic antibody for various solid tumor orcancer in the future.

Anti-Tumor Activity of Bispecific Antibody (In Vivo Model)

The lack of rodent cross reactivity of the PD-L1 and OX40 in bispecificantibodies prevented the use of standard murine syngeneic or humanxenograft tumor models for the assessment of anti-human tumor efficacyof the antibodies. Accordingly, a novel huPBL-SCID-Bg xenogeneic tumormouse model was generated using a SCID-Bg mouse (CB.17/Icr.CgPkrdc^(scid)Lyst^(bg)/CrI), which harbors the beige (Bg) mutation lackmurine T and B lymphocytes and functional NK cells. The anti-human tumorefficacy of the bispecific antibodies was assessed using this model asdescribed below.

The PC-3 human prostate was obtained from American Type CultureCollection and was cultured in RPMI-1640 (Invitrogen) with L-glutamine,sodium pyruvate, penicillin/streptomycin, and 10% heat inactivated fetalbovine serum (FBS, Gibco Cat. No. 10437). Cells were grown to confluencyin T-150 Falcon flasks. Subsequently, cells were trypsinized (Trypsin0.25%-EDTA; Invitrogen) and growth was scaled up to sufficient cellnumber for inoculation. Peripheral blood lymphocytes (PBMCs) wereisolated from heparinized blood using Lymphoprep™ in accordance with themanufactures’ protocol (STEMCELL Technologies Inc.). Counted cellsuspensions were combine such that each mouse received an injection of0.75 × 10⁶ PBMCs and 3 × 10⁶ tumor cells in a single bolus injection of0.1 mL in PBS. In order to facilitate the tumor cells grown in themouse, another 0.1 mL matrigel was then mixed with the combined cellsuspension and then immediately injected into prepare mice.

For each mouse, 0.2 mL volume of the combined cell suspension wasinjected subcutaneously into the right flank of the animal. After 14days inoculation, the solid tumor is formed and reached around 250 to300 mm³ and the bispecific antibody (3 mg/kg of Anti-PD-L1-OX40 Ab-V5),PD-L1 reference antibody (Ref Ab, MPDL3280A) or control antibody(Isotype) is challenged twice per week for three weeks withintraperitoneal injection (i.p.). Tumor measurement was made viaPressier caliper twice per week as well as test sample administrationfor the duration of the experiments and body weights were also recorded.Tumor volume was calculated using the following calculation: length ×width² × 0.44= volume (mm³) and plotted in the FIG. 25 . Mice wereremoved from the study in the event that the tumor volume reached 2000mm³ or animal lost 20% of body weight before termination of theexperiment. Similar results were observed when tumors were measured onday 7 post inoculation, and the animals were randomized according totumor volume. For animal study, each group contained 6 mice. As the datashown in the FIG. 25 , the bispecific antibody showed a significantanti-tumor efficiency in PC-3 xenografted mouse model. The tumor size isshirked at 18 days post tumor inoculation as well as PD-L1 referenceantibody and continued to reduce below 100 mm³. The PC-3 xenograftedmouse model is preliminary demonstrated the anti-tumor of bispecificantibody and revealed its potential to be a therapeutic drug lead in thefuture.

Collectively, these results indicated bi-specific antibody sustain itsimmune checkpoint blocking in PD-1/PD-L1 signaling and agonisticactivity for OX40 signaling. Studies are ongoing to further investigatethe biological activity of these proteins using proper animal model,such as PC-3 tumor in humanized NOD.Cg-Prkdc^(scid) II2rg^(tmlwjl)/SzJ(NSG) model.

The Fc region in the present invention could be from any immunoglobulinisotypes, subclasses, allotypes, or engineered mutants, such as knob andhole Fc fragment(s).

EXAMPLES

The example below describe the generation of monoclonal antibodiessuitable for therapeutic purpose targeting human PD-L1 and OX40.Composite, human anti-human PD-L1 and anti-OX40 antibodies weregenerated from anti-PD-L1 antibody clone 6 and anti-OX40 antibody cloneB17, respectively. Segments of human V region sequence were sourced fromunrelated human antibody (germline and non-germline) sequence databases.

Example 1 Generation of IgG Antibodies That Bind to PD-L1 and OX40

Certain antibodies provided by present invention were originallygenerated from Fabs bind to human PD-L1 or OX40. The Fabs were selectedfrom a phage display library, the OmniMab phagemid library, followingalternating panning on corresponding Fc fusion proteins (PD-L1-Fc orOX40-Fc) and cells expressing human corresponding protein (PD-L1 orOX40). After direct ELISA or cell-based ELISA screening, the positiveclones were then sequenced for heavy chain and light chain. These Fabsincluded those that are designated as “OM-PD-L1-6”, and “OM-PDL1-32”etc. for PD-L1; “OM-OX40-A4”, “OM-OX40-B17”, and “OM-OX40-B19” etc. forOX40. PD-L1 antibodies PD-L1-Clone 3, PD-L1-Clone 6, and PD-L1-Clone 32disclosed in this application were generated from “OM-PD-L1-6” and“OM-PDL1-32”. Meanwhile, OX40 antibodies OX40-A4, OX40-B17, and OX40-B19disclosed in this application were generated from “OM-OX40-A4”,“OM-OX40-B17”, and “OM-OX40-B19” in HEK293 cell or CHO-S cells. Andbispecific antibody targeting PD-L1 and OX40 simultaneously was designedas anti-PD-L1 6-OX40 scFv B17 antibody and anti-PD-L1 6-OX40 scFv B19antibody. The amino acid sequence of the light chain variable region andheavy chain variable region of a given Fab are identical to the aminoacid sequence of the light chain variable region and heavy chainvariable region, respectively.

Example 2 In Vitro Binding of Anti-PD-L1-OX40 scFv to its CorrespondingTarget

Anti-PD-L1-OX40 bispecific antibody was constructed as shown in the FIG.14 and expressed in the HEK293 cells or CHO-S cell. The mediumcontaining bispecific antibody was affinity purified from culturesupernatant by Protein G chromatography. Purified antibody is thenconcentrated, followed by dialysis in PBS buffer and analyzed bySDS-PAGE as shown in the FIG. 15 . To test direct binding of purifiedfusion proteins to PD-L1 or OX40 on ELISA, 100 ng/well recombinant PD-L1or OX40 was coated in a 96-well ELISA plate. Various concentrations ofpurified anti-PD-L1-OX40 scFv were then added to each well and incubatedfor 1 hr. After washing, 1:5000 dilution of anti-Fab HRP conjugate(Jackson Immunochemicals) was added to each well and incubated foranother hour. After final washing, TMB substrate (Invitrogen Inc.) wasadded and OD absorbance at 450 nm was measured. The data analyzed bysigmoidal curve fitting using GraphPad Prism 5 and EC50 is calculated.

Example 3 Antigen Binding Specificity of Anti-PD-L1-OX40 scFv by FACSAnalysis

To test anti-PD-L1-OX40 scFv antibody binding specificity, stable PD-L1expression 293 cells, IFN-γ stimulated A549 or WiDr were stained with 1µg/mL anti-PD-L1-OX40 scFv antibody for 1 hr on ice before wash threetimes with 1x PBS. The bound antibody fusion proteins were detected withAlexa-488 conjugated goat anti-human IgG (H+L) followed by FACSanalysis. Isotype antibody was used as negative control for the test.Results showed anti-PD-L1-OX40 scFv sustains its antigen bindingspecificity as compared with anti-PD-L1 alone. The binding specificityof anti-PD-L1-OX40 scFv antibody was also tested using stable OX40expression 293 cells.

Example 4 In Vitro Immunomodulatory Effect of Bi-Functional Proteins

To measure the ability of the anti-PD-L1-OX40 scFv antibodies tomodulate T cell responsiveness purified T cells will be cultured withallogeneic dendritic cells, prepared by culturing monocytes in GM-CSFand IL-4 for few days. Parallel plates were set up to allow collectionof supernatants at day 3 and day 5 to measure IL-2 and IFN-γrespectivelyusing a commercial ELISA kit. Genentech/Roche’s humanized anti-PD-L1,MPDL3280A will be produced in-house and used as positive control. As thedata shown in the FIGS. 16A and 16B, the IL-2 and IFN-γ production arehighly upregulated in the bispecific antibody treatment as well ascombination treatment after 3 or 5 days antibody treatment. Especially,the bispecific antibody composited by anti-PD-L1 antibody clone 6 andanti-OX40 antibody clone B17 (anti-PD-L1-OX40 scFv B17 Ab) orcombination (anti-PD-L1 clone 6 Ab plus anti-OX40 clone B17 Ab) showedthe enhancement of T cells activation is higher than the combination ofPD-L1 and OX40 reference (PD-L1 Ref Ab plus OX40 Ref Ab). This indicatedthe anti-OX40 B17 antibody may possess a special epitope binding toresult in a better agonistic activity as comparing with reference OX40antibody, GSK3174998.

Example 5 Human Leukocyte Expansion Induced by Bispecific Antibodies InVivo

The lack of detectable cross-reactivity of the PD-L1 or OX40 antibodieswith murine PD-L1 or OX40 and the requirement for the presence of humanimmune cells required the development of models for the in vivofunctional assessment of the bispecific antibodies. Mice with the NODgenetic background carrying the severe combined immunodeficient (SCID)mutation and deficiency in the IL-2 receptor common gamma chain(commonly termed NSG) are able to support the engraftment of largenumber of human peripheral blood leukocytes (huPBL) and maintainengraftment for at least 30 days (King et al., 2008). This mouse model,also known as huPBL-NSG model, was used to assess the functional effectof in vivo systemic administration of the antibodies on human immunecells.

Specifically, 6 million freshly isolated human PBMCs were adoptivelytransferred via intravenous injection into huPBL-NSG mice. Nine dayspost PBMC injections, the animals were administered a single 1 mg/kgdose of mono-antibody, bispecific antibody or IgG4 isotype controlantibody via intraperitoneal injection. At day 24 to 28 post PBMCengraftment, PBMC were stained with antibodies to human and murine CD45assessed via flow cytometry. Forward and side scatter profiles were usedto determine a lymphocyte gate. Bispecific antibodies were able toenhance expansion of human leukocytes as evidenced by increasedproportion of human CD45+ cells in the peripheral blood of engraftedmice. For each group, n≧6 mice.

Example 6 Inhibition of PC-3 or A498 Tumor Cell Growth in huPBL-NSG byAnti-PD-L1-OX40 scFv Antibody

PD-L1 positive human prostate cancer cell line, PC-3 (ATCC^(#) CRL-1435)or kidney cancer cell line, A498 (ATCC® HTB-44™) can be used toestablish xenograft models in huPBL-NSG mice. For tumor formation, 3 ×10⁶ PC-3 cells (or A498 cells) /mouse will be injected subcutaneously inhuPBL-NSG mice as described above. In order to assess the inhibitoryeffects on the tumor growth, different concentrations of anti-PD-L1-OX40scFv antibody, reference antibody, or isotype antibody from 0.1-3 mg/kgwill be administered intravenously twice weekly for 4 weeks in the miceafter 14 days tumor cells implantation. The tumor growth will bemeasured twice per week up to 5 weeks as described in the Fox ChaseSCID®Beige mice model.

Example 7 Pharmacokinetic Assessment of anti-PD-L1-OX40 scFv in Mice andMonkeys

10-40 mg / kg of bi-functional proteins, anti-PD-L1-OX40 scFv will beadministered into mice or monkeys via subcutaneous injection orintravenous injection. Serum samples will be taken at different timepoints after the injection up to 15 days. Concentrations of the Fcfusion protein in the serum samples will be determined using asandwiched ELISA assay.

Although the present invention has been described in considerable detailwith reference to certain embodiments thereof, other embodiments arepossible. Therefore, the spirit and scope of the appended claims shouldnot be limited to the description of the embodiments contained herein.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentinvention without departing from the scope or spirit of the invention.In view of the foregoing, it is intended that the present inventioncover modifications and variations of this invention provided they fallwithin the scope of the following claims.

What is claimed is:
 1. An antibody or an antigen-binding portion thereofbinding to OX40 (CD134), comprising: a heavy chain variable region and alight chain variable region respectively comprising an amino acidsequence of at least 90% sequence identity to the amino acid sequenceof: SEQ ID NO: 8 and amino acid 1-108 of SEQ ID NO: 7, or amino acid128-246 of SEQ ID NO: 10 and amino acid 1-112 of SEQ ID NO: 10, whereinthe at least 90% sequence identity to the amino acid sequence excludescomplementarity-determining regions (CDRs).
 2. The antibody or theantigen-binding portion thereof of claim 1, wherein the antibody or theantigen-binding portion thereof is a single chain variable fragment(scFv) sequence selected from the group consisting of SEQ ID NO: 10, and12.
 3. The antibody or the antigen-binding portion thereof of claim 1,wherein the antibody or the antigen-binding portion thereof is abispecific antibody.
 4. The antibody or the antigen-binding portionthereof of claim 3, wherein the bispecific antibody comprises an immunecheckpoint protein binding site.
 5. The antibody or the antigen-bindingportion thereof of claim 4, wherein the immune checkpoint proteinbinding site comprises a programmed cell death protein 1 ligand (PD-L1)binding site, PD-1 binding site, epidermal growth factor receptor (EGFR)binding site, human epidermal growth factor receptor 2 (HER2) bindingsite, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) binding site,or lymphocyte activation gene 3 (LAG3) binding site.
 6. An antibody oran antigen-binding portion thereof binding to PD-L1, comprising: a heavychain variable region and a light chain variable region respectivelycomprising an amino acid sequence of at least 90% sequence identity tothe amino acid sequence of SEQ ID NO: 4 and amino acid 1-110 of SEQ IDNO: 3, wherein the at least 90% sequence identity to the amino acidsequence excludes CDRs.
 7. A bispecific antibody comprising at least oneof polypeptide chain, wherein the polypeptide chain comprises: an OX40binding site, comprising: a heavy chain variable region and a lightchain variable region respectively comprising an amino acid sequence ofat least 90% sequence identity to the amino acid sequence of amino acid128-246 of SEQ ID NO: 10 and amino acid 1-112 of SEQ ID NO: 10, whereinthe at least 90% sequence identity to the amino acid sequence excludesCDRs; and a PD-L1 binding site, comprising: a heavy chain variableregion and a light chain variable region respectively comprising anamino acid sequence of at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 2 and amino acid 1-111 of SEQ ID NO:
 1. 8. Thebispecific antibody of claim 7, wherein the polypeptide chain furthercomprises: a Fc domain; a Fab fragment connected to the N-terminus ofthe Fc domain, and the Fab fragment comprising the PD-L1 binding site;and a scFv connected to the C-terminus of the Fc domain, and the scFvcomprising the OX40 binding site.
 9. The bispecific antibody of claim 8,wherein the polypeptide chain further comprises a linker between the Fcdomain and the scFv.
 10. The bispecific antibody of claim 7, wherein thebispecific antibody comprises one pair of polypeptide chains.
 11. Thebispecific antibody of claim 10, wherein the bispecific antibody is anIgG, IgE, IgM, IgD, IgA, or IgY antibody.
 12. The bispecific antibody ofclaim 11, wherein the bispecific antibody is an IgG antibody.
 13. Thebispecific antibody of claim 12, wherein the IgG antibody is an IgG1,IgG2, IgG3, or IgG4 antibody.
 14. An antibody-drug conjugate comprising:a therapeutic agent; and an antibody or an antigen-binding portionthereof binding PD-L1 and/or OX40, wherein the therapeutic agent iscovalently conjugated to the antibody or the antigen-binding portionthereof by a linker; wherein the antibody or the antigen-binding portionthereof is: a) an antibody or an antigen-binding portion thereof bindingto OX40, comprising: a heavy chain variable region and a light chainvariable region respectively comprising an amino acid sequence of atleast 90% sequence identity to the amino acid sequence of: SEQ ID NO: 8and amino acid 1-108 of SEQ ID NO: 7, or amino acid 128-246 of SEQ IDNO: 10 and amino acid 1-112 of SEQ ID NO: 10, wherein the at least 90%sequence identity to the amino acid sequence excludes CDRs; b) anantibody or an antigen-binding portion thereof binding to PD-L1,comprising: a heavy chain variable region and a light chain variableregion respectively comprising an amino acid sequence of at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 4 and aminoacid 1-110 of SEQ ID NO: 3, wherein the at least 90% sequence identityto the amino acid sequence excludes CDRs; or c) a bispecific antibodycomprising at least one of polypeptide chain, wherein the polypeptidechain comprises: an OX40 binding site, comprising: a heavy chainvariable region and a light chain variable region respectivelycomprising an amino acid sequence of at least 90% sequence identity tothe amino acid sequence of amino acid 128-246 of SEQ ID NO: 10 and aminoacid 1-112 of SEQ ID NO: 10, wherein the at least 90% sequence identityto the amino acid sequence excludes CDRs; and a PD-L1 binding site,comprising: a heavy chain variable region and a light chain variableregion respectively comprising an amino acid sequence of at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 2 and aminoacid 1-111 of SEQ ID NO: 1, wherein the at least 90% sequence identityto the amino acid sequence excludes CDR.
 15. A pharmaceuticalcomposition comprising the antibody or the antigen-binding portionthereof according to claim 1, and at least one pharmaceuticallyacceptable carrier.
 16. A pharmaceutical composition comprising theantibody or the antigen-binding portion thereof as claimed in claim 6,and at least one pharmaceutically acceptable carrier.
 17. Apharmaceutical composition comprising the bispecific antibody accordingto claim 7, and at least one pharmaceutically acceptable carrier.
 18. Amethod of treating cancer comprising administering to the subject inneed thereof an effective amount of the antibody or antigen-bindingportion thereof according to claim
 1. 19. The method of claim 18,wherein the cancer is selected from the group consisting of prostatecancer, lung cancer, Non-Small Cell Lung Cancer (NSCLC), melanoma,lymphoma, breast cancer, head and neck cancer, renal cell carcinoma(RCC), and ovarian cancer.
 20. A method of treating cancer comprisingadministering to the subject in need thereof an effective amount of theantibody or antigen-binding portion thereof as claimed in claim
 6. 21.The method of claim 20, wherein the cancer is selected from the groupconsisting of prostate cancer, lung cancer, Non-Small Cell Lung Cancer(NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renalcell carcinoma (RCC), and ovarian cancer.
 22. A method of treatingcancer comprising administering to the subject in need thereof aneffective amount of the bispecific antibody according to claim
 7. 23.The method of claim 22, wherein the cancer is selected from the groupconsisting of prostate cancer, lung cancer, Non-Small Cell Lung Cancer(NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renalcell carcinoma (RCC), and ovarian cancer.
 24. A nucleic acid moleculeencoding the antibody or the antigen-binding portion thereof accordingto claim
 1. 25. A nucleic acid molecule encoding the antibody or theantigen-binding portion thereof according to claim
 6. 26. A nucleic acidmolecule encoding the bispecific antibody according to claim 7.